Genetic and Molecular Properties of an Infectious Antibiotic Resistance (R) Factor Isolated from Klebsiella

A Klebsiella strain of human origin that was resistant to ampicillin, chloramphenicol, kanamycin, neomycin, streptomycin, and tetracycline was found to have all of these resistances associated with a R factor and a satellite molecular species of deoxyribonucleic acid (DNA) with an average buoyant density of 1.710 in cesium chloride gradients. There was no evidence of the existence of DNA with other buoyant densities. The strain bears two separable mutations for chloramphenicol resistance, both of which are associated with the R factor (KR9). Exposure of the Klebsiella strain to acridine derivatives or to ethidium bromide (which was more efficient) resulted in partial losses of resistance accompanied by the disappearance of the satellite DNA peak or shifts in its density. The R factor and its component genes were conjugally transmitted across generic boundaries and maintained in new hosts with different efficiencies. The basis of this difference lies not only in the efficiency of conjugal transfer but also in the stability of the components after transfer. All of the resistance genes and the resistance transfer factor were cotransducible by phage Plkc from Escherichia coli. Partially resistant strains could be reconstituted to full resistance or to a recombined pattern of partial resistance by conjugation with donors having complementary resistance patterns. This recombination serves as an efficient mechanism for rescuing superinfecting genes that are otherwise intracellularly excluded. KR9 is an fi⁺ type of R factor which in the natural state does not appear to be as repressed in conjugal transfer as other R factors.     

The fate of yeast mitochondrial DNA after ultraviolet irradiation. I. Degradation during post-UV dark liquid holding in non-nutrient medium.

A partial recovery of ultraviolet (U.V., 254 nm) induced petite mutation (?^?) is observed in exponential phase yeast. This process requires a period of dark holding (LH) in non-nutrient medium followed by growth in nutrient medium. At intervals during LH prelabelled DNA was examined by equilibrium cesium chloride gradients. The gradual decrease in ?^? was accompanied by an ongoing degradation of mitochondrial DNA (mitDNA) during the first 24 hours followed by a stabilization. The dose response for mitDNA degradation was biphasic. No new synthesis of mitDNA occured during LH. MitDNA remaiting after degradation showed a) slight shift to a heavier buoyant density indicating a possible selective degradation of A-T regions b) no difference in size when compared to non-irradiated samples. The first step in the recovery of the ?^? mutation is mitDNA degradation followed by other events taking place when growth resumes.     

Characterization of DNA components from some colorless algae

The DNA components of five colorless algae were characterized by their buoyant densities in cesium chloride. Two DNA components were detected in Polytoma obtusum and Polytoma uvella. Upon renaturation of the thermally denatured DNA the minor and approx. 15% of the major DNA component returned to their native densities. The buoyant densities of the major and minor DNA of P. obtusum and P. uvella are different from that of the morphologically and biochemically similar green alga Chlamydomonas reinhardtii. A major and a minor DNA component with the same buoyant densities as that of the green alga Euglena were also found in Astasia longa, which is morphologically similar to Euglena. The renaturation of the minor but not the major component was readily detectable by the change in buoyant density. Only one DNA component was detected in Polytomella agilis and Polytomella caeca. After thermal denaturation approx. 5% of each of these DNA components were renatured readily. Based on these data, the possible evolutionary origin of these colorless algae is discussed.     

Reciprocal induction of cell division by cells of complementary mating types in Tetrahymena

Chick embryo fibroblasts in monolayer culture were synchronized by contact inhibition and serum starvation. Nuclear DNA isolated from the [³H] thymidine pulse-labelled cells throughout the period of DNA synthesis (S phase) was analysed by hydroxylapatite chromatography after renaturation at different C₀t values. It is shown that repeated sequences having different frequencies of reassociation, replicate differently throughout the S period. In order to study the distribution of the repeated sequences, DNA isolated during the S period was fractionated according to its buoyant density. It is shown that only some of the highly reiterated sequences which are included in the high buoyant density DNA fractions, replicate equally well during the early and the late S periods. By contrast, reiterated sequences of the low buoyant density DNA fractions replicate mainly during the late S period.     

ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL DNA FROM DROSOPHILA MELANOGASTER

Mitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm³ has been isolated from unfertilized eggs of Drosophila melanogaster. This DNA is a circular molecule with an average length of 5.3 µm; it reassociates with a low C₀t_1/2 after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm³. MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm³, slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function.     

Physical Heterogeneity Among Bacillus subtilis Deoxyribonucleic Acid Molecules Carrying Particular Genetic Markers

In a Bacillus subtilis deoxyribonucleic acid (DNA) preparation, extracted and purified by the Marmur procedure, the DNA molecules carrying a particular marker are heterogeneous with respect to molecular weight, buoyant density, and thermal stability. This finding constitutes evidence against unique points of breakage during DNA isolation. The variation in buoyant density suggests a local compositional heterogeneity in the chromosomal region of certain markers. The variation in molecular weight provides an explanation for the results of certain transformation experiments that are otherwise poorly understood. An example of such a result is the observation that acridine orange increases the efficiency of differential thermal inactivation of markers. An explanation of this phenomenon is suggested by the demonstration that acridine orange can decrease the natural intramarker heterogeneity in melting behavior.     

Effect of L-phenylalanine on I-methyladenine production and spontaneous oocyte maturation in starfish

5-Bromodeoxyuridine (BUdR)-resistant cells were obtained from N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-treated soybean protoplasts and cultured in liquid nutrient medium containing BUdR (20 μg/ml) and uridine (100 μg/ml). Addition of uridine to the medium improved growth of the BUdR-resistant cells. The growth of BUdR-resistant cells was partly inhibited when hypoxanthine, aminopterine, glycine and thymidine were added to the medium. Both BUdR-resistant and BUdR-sensitive cells exhibited thymidine kinase activity. CsCl density gradient analyses showed that the DNA of BUdR-resistant cells, which were cultured in the presence of BUdR, had a buoyant density of 1.703 g/ml, while the DNA of the parental soybean cells grown without BUdR had a buoyant density of 1.692 g/ml. Uptake of ³H-thymidine or ¹⁴C-BUdR by the cells occurred in both BUdR-resistant and BUdR-sensitive cells. CsCl density gradient patterns of labelled DNA also demonstrated that ¹⁴C-BUdR and ³H-thymidine were incorporated into the DNA of BUdR-resistant cells, as well as into that of BUdR-sensitive cells.     

Transformation in Bacillus subtilis: characterization of an intermediate in recombination observed during transformation.

Summary From recombination-proficient competent cells of Bacillus subtilis in which the donor DNA entered at 17°, and which were kept at the same temperature, a complex of donor DNA and the recipient chromosome can be obtained which has a relatively high buoyant density in CsCl gradients. Exposure of the isolated complex to nuclease S1 liberates donor radioactivity. The limited biological activity of DNA re-extracted from cells attempting to recombine at 17° is decreased upon incubation with nuclease S1. If recombination is allowed to proceed at 30°, the high buoyant density of the donor-recipient complex decreases to normal values and less radioactivity can be liberated from the complex by nuclease S1. Concomitantly the biological activity of re-extracted DNA becomes less vulnerable to nuclease S1 under these conditions. On the basis of these observations we assume that the intermediate complex partly consists of unpaired single-stranded donor DNA.Support for the correctness of this assumption is derived from experiments with a mutant, which is delayed in the processing of high buoyant density donor-recipient complex to normal buoyant density donor-recipient complex. This delay is reflected in the time of acquisition of resistance to nuclease S1 digestion of the isolated complex.     

The ribosomal cistrons of the water mold Achlya bisexualis

Total DNA was extracted from vegatative mycelia of the water mold Achlya bisexualis. Fractionation of the DNA in CsCl gradients resulted in two components: a major component with a buoyant density of 1.697 g cm^?3 and a minor component with a density of 1.685 g cm^?3. The minor component has been identified as mitochondrial DNA based on extractions from isolated mitochondria and Triton X-100 washed nuclei. Detergent washing of the nuclei yielded DNA in which the mitochondrial DNA component was absent, while the isolated mitochondrial preparations contained DNA enriched in the 1.685 g cm^?3 component. Hybridization studies of A. bisexualis DNA to rRNA show that the ribosomal cistrons have a buoyant density coincident with that obtained with the nuclear DNA. In addition, preliminary evidence indicates that the mitochondrial DNA does not hybridize to the cytoplasmic RNA under the conditions used for this study. Ribosomal RNA hybridized to about 0.65% of the total DNA.     

Characterization of the most rapidly renaturing sequences in mouse main-band DNA

The most rapidly renaturing sequences in the main-band DNA of Mus musculus, isolated on hydroxyapatite, are found to consist of two discrete families: a presumed “foldback” DNA fraction and a fraction renaturing bimolecularly. The latter family, which we call “main-band hydroxyapatite-isolated rapidly renaturing DNA”, has a kinetic complexity about an order of magnitude greater than that of mouse satellite DNA. It shows about twice as much mismatching as renatured mouse satellite, as judged by its thermal denaturation curve. In situ hybridization localizes the sequences to all chromosomes in the mouse karyotype, and to at least several regions of each chromosome. The in situ result and solution hybridization studies eliminate the possibility that the main-band rapidly renaturing DNA is composed of mouse satellite sequences attached to sequences of higher buoyant density. Nuelease S₁ digestion experiments disclose that even at low molecular weight there are unrenatured “tails” attached to the rapidly renaturing sequences. When the main-band DNA fragment size is increased the amount of rapidly renaturing sequences remains constant, but the amount of attached tails of unrenatured DNA increases as judged by S₁ nuclease digestibility, hyperchromicity and buoyant density. It is concluded that at least 5% of the mouse genome is composed of segments of the rapidly renaturing sequences averaging about 1500 base pairs, alternating with segments of more complex DNA averaging about 2200 base pairs. This interspersion of sequences is compared to that found in several other organisms. The properties of the foldback DNA are similarly investigated as a function of DNA fragment size.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.     

A revised equation relating DNA buoyant density to guanine plus cytosine content

An equation relating DNA buoyant density of CsCl to G + C content is given which uses the correct density value of Escherichia coli DNA as the reference. This is done to eliminate the current confusion brought about by two references states.     

Isolation and partial characterization of a mitochondrial deoxyribonucleic acid-protein complex from sea urchin embryos

Summary Lysis of mitochondria from sea urchin embryos with Triton X-100 led to a complete conversion of DNA-containing mitochondrial residues into protein-DNA complex with a density higher than 1.22 g/cm³ in sucrose solutions. This complex banded isopycnically in metrizamide gradients at a density of 1..26 g/cm³. Exposure to mixtures of Triton X-100 with Tween 80 resulted in progressively less delipitated and disorganized mitochondria over Tween/Triton weight ratios from 1 to 2, with the retention of the starting buoyant density in sucrose of approximately 1.16 g/cm³ at Tween/Triton ratios above 2.5. The DNA-internal protein complex sedimented with the bulk of the surviving mitochondrial structure under all conditions studied. No free DNA could be detected under any conditions of membrane removal.     

Resolution of α, β and γ DNA of Saccharomyces cerevisiae with the antitumor drug cis-Pt(NH3)2Cl2. Evidence for preferential drug binding by GpG sequences of DNA

This paper reports further studies on the separation of DNAs with the antitumor drug cis-Pt(NH₃)₂Cl₂. cis-Pt(NH₃)₂Cl₂ permits resolution of the three DNA components from whole Saccharomyces cerevisiae in CsCl gradients, avoids pelleting of mitochondrial (β) DNA and does not require a critical molar ratio of platinum drug to DNA-P. However, the difficulty in removing all of the DNA-bound platinum may limit its preparative use. The linear relationship between the increase in buoyant density of platinized double-stranded DNA and its G + C content is employed to calculate a G + C content of 41.2% and 45.8% for α and γ DNA, respectively, using a value of 20% G + C for β DNA. In parallel experiments, we find that poly(dG)·poly(dC), which contains sequential guanine bases, exhibits an unexpectedly large buoyant density increase with cis-Pt(NH₃)₂ Cl₂, while the buoyant density increase of poly[d(G-C)]is markedly retarded, indicating an effect of nucleotide base sequence on DNA separation. The trans platinum compound, which has no antitumor properties, separates DNAs on the basis of G + C content in a similar fashion, but does not preferentially increase the buoyant density of poly(dG)·poly(dC).     

Genome complexity of the maize rust fungus,Puccinia sorghi

The base composition and complexity of genomic DNA fromPuccinia sorghi have been estimated by thermal denaturation, analytical ultracentrifugation, and reassociation kinetics. The buoyant density of genomic DNA in CsCl was found to be 1.7021 g/ml, which corresponds to a GC content of 43%. From thermal denaturation curves the GC content was estimated to be 41%. The haploid genome size ofP. sorghi was estimated to be 4.7 × 10⁷ bp, half of which represented a moderately repetitive fraction. The size of theP. sorghi genome is similar to that of other basidiomycete fungi; however, the amount of repetitive DNA is greater than that reported for most other fungi.     

A genetic and biochemical analysis of petite mutations in yeast

A series of spontaneous cytoplasmic petite mutants was isolated from a grande strain of Saccharomyces cerevisiae doubly marked with the cytoplasmically inherited determinants to erythromycin and oligomycin resistance. The petites were characterized with regard to the genetic stability of these antibiotic resistance markers and to their degree of suppressivity. No relation was found between the genetic instability of a petite mutant and the degree of suppressivity exhibited by that mutant. Three petites of 19.4%, 57.4% and 90.4% suppressivity were selected and their mitochondrial DNA characterized with regard to molecular weight, buoyant density in analytical cesium chloride density gradients, and the percentage of the total cellular DNA represented by the mitochondrial DNA. From these results it appears that the molecular weight of the mitochondrial DNA of the petite strains examined is the same as that shown by the parental grande strain, regardless of the degree of suppressivity exhibited.     

Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis

Euglena chloroplast DNA has a buoyant density in CsCI of 1.686. Shearing this DNA produces a satellite band at density 1.700. The satellite, easily lost during preparative CsCI gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 × 10⁶ daltons of DNA.     

Characterization of DNA from Rhizobium cells and their bacteriods from root nodules

DNA from two rhizobial strains $\text{Cicer}$ and $\text{Phaseolus}$ and their bacteroids from root nodules have been isolated, purified and characterized for thermal denaturation temperature and buoyant density. Bacteroid DNA had a lower Tm value and buoyant density comparad to $\text{Rhizobium}$ cell DNA. The calculated GC content of becteroid DNA was lower than the $\text{Rhizobium}$ cell DNA.     

Transformation and Physical Properties of R-Factor RP4 Transferred from Escherichia coli to Rhizobium trifolii

Transformation of R-factor RP4 specifying resistance to ampicillin, kanamycin, and tetracycline from Escherichia coli to Rhizobium trifolii is reported. Partially purified RP4 deoxyribonucleic acid (DNA) of the donor strain E. coli J5-3 that carried the R-factor was prepared by the lysozyme-ethylenediaminetetraacetic acid-Triton X-100 procedure and was used in transformation experiments with R. trifolii as recipient. The frequency of transformation of the R-factor into R. trifolii was 1.3 × 10⁻⁴. Dye buoyant density and sucrose gradient centrifugation of R. trifolii DNA showed that the expression of the specified drug resistance of RP4 by R. trifolii was accompanied by the acquisition of an extrachromosomal, satellite DNA component which has indistinguishable physical properties from the R-factor in the donor strain. The significance of the transformation is discussed.